Primers for Respiratory Virus Diagnostics

Several viruses can infect the lower and upper respiratory tract of us humans. The respiratory system allows us to breathe. The respiratory system includes several organs and structures needed to exchange gases such as oxygen (in) and carbon dioxide (out). Parts of the respiratory system are the nose and nasal cavity, the sinuses, mouth, throat, voice box, windpipe, diaphragm, and the lungs. Many common viral infections target the upper respiratory system causing severe symptoms in infants, the elderly, and patients with lung or heart problems.

The list of common respiratory viruses includes the epidemic influenza viruses A, B, C, avian influenza viruses, parainfluenza viruses 1–4, adenoviruses, coronaviruses, orthohantaviruses, and respiratory syncytial virus and human metapneumovirus, as well as rhinoviruses.

Symptoms of different respiratory infections, also known as clinical presentation, caused by various viral pathogens, can be very similar. Hence, the correct diagnosis is quite tricky. A rapid virological method will allow a specific and sensitive diagnosis at an early stage of the infection. Significant advances in modern molecular technics have enabled the speedy and sensitive detection of viral pathogens. Polymerase chain reaction (PCR) based methods are now considered as the gold standard of viral assays. For many RNA viruses, including respiratory viruses, multiplex reverse transcription (RT)-PCR assay-based diagnosis allows rapid, sensitive, and specific detection.

PCR primers and probes for respiratory syncytial viruses A and B and parainfluenzavirus 3, causing bronchiolitis in children.

Eugene-Ruellan et al., in 1998, developed a reverse-transcription-PCR and hybridization-enzyme immunoassay (RT-PCR-EIA) for the detection and identification of significant bronchiolitis agents in infants. Bronchiolitis is a common lung infection in young children and infants. Bronchiolitis refers to inflammation and congestion in the small airways, also known as bronchioles of the lung. In children and infants, the respiratory syncytial viruses A and B (RSVA and RSVB) and parainfluenzavirus 3 (PIV3) cause bronchiolitis of the lower respiratory tract.

For virus diagnosis, the research group designed two primer sets (P1-P2 and P1-P3) using sequences in the polymerase L gene's conserved region. This molecular method allows detection in a single step and paramyxoviruses RSVA, RSVB, and PIV3 typing. Please review the alignment of the functional motifs A and C in the viral L gene in Eugene-Ruellan et al.

Table 1: PCR primers and probes for respiratory syncytial viruses A and B and parainfluenzavirus 3.

Primers for influenza viruses, parainfluenza viruses, adenoviruses, coronaviruses, orthohantaviruses, respiratory syncytial virus and human metapneumovirus, and rhinoviruses.

Coiras et al., in 2004, developed a multiplex RT-nested PCR assay for the detection and identification of several respiratory viruses.These include the human parainfluenza viruses types 1, 2, 3, and 4AB, the coronaviruses type 229E and OC43, and generic human enteroviruses and rhinoviruses. The researchers designed primers selecting sequences from the conserved regions of haemagglutinin genes, the conserved regions of coronavirus spike protein genes, and the polyprotein gene of rhinoviruses and enteroviruses, between the 5’-non-coding region (5’-NCR) and VP4/VP2 regions. Table 1 lists GenBank accession numbers of the viral sequences,  sequences, and properties of all primers studied.

^a1, forward;2,reverse in first-round RT-PCR. ^b3, forward;4,reverse in nested PCR. ^cPrimer located up-stream from coding region for haemagglutinin gene. ^dGene position referred to Poliovirus1strain Sabin (Accession no. V01150). Note: All rhinoviruses have a deletion of approximately 116 bp as regards enteroviruses.

Gunson et al., in 2005, described a real-time RT-PCR multiplex assay for the detection of 12 respiratory viral infections using a triplex reaction. Specific labeled probes enabled the convenient interpretation of results as generated by the multiplex format.

Table 3: Primers and probes used in triplex real-time RT-PCR assays (Adapted from Gunson et al. in 2005).

Van de Pol et al., in 2007, used primers for real-time PCR diagnostic of respiratory viruses from patients admitted with respiratory symptoms. Diagnostic of specific respiratory viruses allows clinicians to initiate optimal patient management and initiate adequate (future) use of antiviral therapy and optimal infection control.

 ^aFAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; MGB, minor groove binding.

Sequence variations in probe binding sites affect detection of respiratory syncytial virus group B by real-time RT-PCR.

Kamau et al., in 2017, reported that direct immunofluorescence tests (IFATs) and multiplex real-time RT-PCR assays revealed discrepancies in observing an increasing number of RSV-B viruses not detected by PCR. Mismatches in primer and probe binding sites revealed by sequencing the nucleoprotein (N protein) and glycoprotein (G protein) genes were the cause. The researchers detected three (3) different mismatches in the probe-target sites of viruses not seen via PCR. These results allowed the research group to design new primers and probes.

Table 5: Primer and probes for the old and the new N rt-RT-PCR assays.

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Primer and Probes!

Reference

Bronchiolitis

M.T. Coiras, J.C. Aguilar, M.L. García, I. Casas, and P. Pérez-Breňa; Simultaneous Detection of Fourteen Respiratory Viruses in Clinical Specimens by Two Multiplex Reverse Transcription Nested-PCR Assays. Journal of Medical Virology 72:484–495 (2004). [PMC]

Eugene-Ruellan G, Freymuth F, Bahloul C, Badrane H, Vabret A, Tordo N. Detection of respiratory syncytial virus A and B and parainfluenzavirus 3 sequences in respiratory tracts of infants by a single PCR with primers targeted to the L-polymerase gene and differential hybridization. J Clin Microbiol. 1998 Mar;36(3):796-801. [PMC]

Gunson RN, Collins TC, Carman WF. Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions. J Clin Virol. 2005 Aug;33(4):341-4. [PMC]

Infectious Diseases

Kamau E, Agoti CN, Lewa CS, Oketch J, Owor BE, Otieno GP, Bett A, Cane PA, Nokes DJ. Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR. J Clin Virol. 2017 Mar; 88:21-25. [PMC]

Poch O, Sauvaget I, Delarue M, Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding element. EMBO J. 1989;8:3867–3874. [PMC]

The Respiratory System

Alma C. van de Pol, Anton M. van Loon, Tom F. W. Wolfs, Nicolaas J. G. Jansen, Monique Nijhuis, Els Klein Breteler,1 Rob Schuurman, and John W. A. Rossen;  Increased Detection of Respiratory Syncytial Virus, Influenza Viruses, Parainfluenza Viruses, and Adenoviruses with Real-Time PCR in Samples from Patients with Respiratory Symptoms. JOURNAL OF CLINICAL MICROBIOLOGY, July 2007, p. 2260–2262. [PMC]

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